The only licensed human anthrax vaccine, anthrax vaccine absorbed (AVA), was developed in the late 1950s and is poorly immunogenic. The prolonged 18-month vaccination regimen and required annual boosters are especially problematic for immunization of military personnel both in terms of safety and in terms of practicality. The possibility of new virulent strains that have been strategically engineered to subvert the limited immune response elicited by the AVA vaccine constitutes a genuine threat. Cerus has developed a novel approach for clinically safe and potent vaccines against microbial pathogens utilizing its technology based on the proprietary S-59 psoralen. The S-59 psoralen (S-59) is approved for human use in Europe as part of the commercially available INTERCEPT pathogen inactivation system for platelets. As a proof of concept, mutant strains of the human pathogen Listeria monocytogenes were created by deleting the uvrAB genes. This deletion rendered the DNA repair mutant bacteria exquisitely sensitive to S-59/UVA light-mediated inactivation, but preserved metabolic activity and expression of the Listeria genetic repertoire. As a result, S-59/UVA-inactivated Listeria uvrAB-induced protective memory T cell responses and significant antibody responses in vaccinated animals. With this application, we propose to construct a panel of S-59/UVA inactivated anthrax vaccine candidates based on nonsporogenic B. anthracis DNA repair mutants utilizing host strains having both pXO1 and pXO2 virulence plasmids. The primary goal of this proposal is to identify a vaccine candidate for further testing in nonhuman primates. A series of experiments to be performed in both mice and guinea pigs are proposed to identify an optimal anthrax vaccine candidate based on safety, combined with immunogenicity and induction of protection against lethal toxin or spore challenge. We hypothesize that this unique approach of combining non-viability with metabolic activity within the context of the whole B. anthracis organism will result in a vaccine that induces protective bacterial-specific immunity with increased depth (i.e. mucosal, humoral, and cellular immunity), breadth (immune response targeted at multiple bacterial antigens including capsule), and durability (long-term immunological memory) with a practical immunization regimen, as compared to the AVA vaccine. Achieving the primary goal set forth in this proposal will set the stage for longer-term objectives of the program, and for moving the project toward an initial human clinical trial to test the immunogenicity and safety of this platform. The ultimate goal of this work is to replace the current AVA human anthrax vaccine with an S-59 psoralen/UVA light inactivated genetically defined attenuated nonsporogenic strain of B. anthracis. To accomplish this goal to develop what is a new class of vaccines that combine the safety of a killed vaccine with the potency of a live-attenuated vaccine, we have assembled an experienced consortium comprised of scientists with documented expertise in bacterial pathogenesis, Gram-positive genetics, novel vaccine platform development, infectious disease vaccine development, and a history of productive collaboration.